IE46795B1 - Hepatitis b core antigen - Google Patents

Description

This invention relates to hepatitis B core antigen (HBcAg).

Hepatitis B is one of the types of viral hepatitis which results in a systemic infection with the principal pathologic changes occurring in the liver . This disease affects mainly adults and is maintained chiefly by transfer of infection from long-term carriers of the virus. Usual methods of spread are by blood transfusion, contaminated needles and syringes, through skin breached by cuts or scratches, by unsterilized dental instruments as well as by saliva, veneral contact or exposure to aerosolized infected blood.

The incubation period of type B hepatitis is relatively long: from 6 weeks to 6 months may elapse between infection and the onset of clinical symptoms. The illness usually begins with fatigue and anorexia, sometimes accompanied by myalgia and abdominal discomfort.

Later jaundice, dark urine, light stools and tender hepatomegaly may appear . In some cases, the onset may be rapid, with appearance of jaundice early in association with fever, chills, and leukocytosis. In other cases jaundice may never be recognized and the patient may be aware of a flu-like illness. It is estimated that the majority of hepatitis infections result in a mild, anicteric illness. .46795 - 3 Serum obtained from patients with hepatitis B infections usually contains three distinct morphologic forms. The largest of these morphologic forms, a 42-nm to 45-nm double shelled spherical particle, often referred to as the Dane particle (HBV), is believed to be the virus of hepatitis B. The outer surface or envelope of the Dane particle (HB Ag) surrounds a 27-nm inner core which does not react with antibody against HB^Ag and which contains a distinct antigen, the core antigen (HB^Ag). Antibody to HBcAg appears after acute hepatitis B infection, and also can be demonstrated consistently in chronic carriers of HB^Ag. Highly sensitive techniques are now available for detection of the HB^Ag system. A deterrent to the more widespread use of such techniques, however, is the absence of a simple yet practical and effective method for obtaining HB^Ag. The methods proposed heretofore generally involve the use of selected plasma which contains exceptionally high amounts of Dane particles.

The preparation of HB^Ag is isopycnic banding of biological fluid from human HBgAg positive donors, optionally but preferably pelleting the Dane particles, and then removing the surface antigen by contacting the Dane particles with a nonionic surfactant having from 15 to 35 oxyethylene units in the presence of a reducing agent such as mercaptoethanol, is described and claimed in the specification of Patent Specification No. 46793 Attention is also directed to the specification of Patent Specification No. 46794 The present invention provides a composition comprising hepatitis B core antigen in a medium consisting essentially of TMN-1% BSA as hereinafter defined, saline or carbonate buffer, e.g. SPGA. Also in accordance with - 4 4679 5 the present invention, such a composition is inactivated to produce a vaccine effective to produce hepatitis B core antibody, which is another embodiment of the present invention. Inactivation may be effected by an aqueous solution of formaldehyde and the core antigen may be adsorbed on a physiologically acceptable substrate, e.g. alum.

In the following Examples which illustrate the invention, all percentages are by weight, the term IAHA means immune adherence agglutination assay and SPGA is a stabilizer composed of sucrose, phosphate, glutamine and albumin disclosed by Bovarnick et al, J. Baet. 59 (1950) pp 509-521.

Example 1 Dane particles are prepared and purified by the method of Example 1 of the specification of Patent Specification No. 46793 . Then the surface antigen is removed as described in that Example by treatment with 2-mercaptoethanol and polyoxyethylene (20) sorbitan monooleate. The resulting mixture is agitated gently and placed in a 37°C water bath. After 1 hour the mixture is diluted with TMN-1% BSA (which means a solution containing 0.08 M Tris, 0.008 M MgCl^ and 0.14 M Ha Cl, and l/ί BSA) using a previously calculated quantity of diluent until it contains 32 IAHA units per ml. The solution is then dispensed into plastic 2-ml screw-cap serum tubes (0.5 ml/tube) arid stored in a liquid-nitrogen freezer.

Example 2 HB^Ag prepared as in Example 1 of Patent Specification No. 46793 is adsorbed on alum as follows. 10 ml of HB Ag (Type Ad) containing 16-32 IA units/ml is mixed - 5 I 6 7 9 5 with 0.85 ml of 10% of alum solution KAI(S04)2.12H2O.

While stirring 0.1 N NaOH is added slowly to adjust the pH to 6.8. Mixing is continued for 1 hour at room temperature. The solution is then centrifuged at 1500 x g for 10 minutes. The supernate is decanted and the pellet is resuspended with saline solution to the original volume (10 mis). The solution is then mixed for 5-10 minutes prior to use as an antigen. This procedure is repeated but using 10 ml of HB^Ag (type Ay) instead of HBcAg (type Ad).

Example 3 The final product of Example 1 is treated under aseptic conditions with 1:4000 formalin at 37°C for 72 hours. Excess of formalin is then neutralized with sodium bisulfite. The core antigen is then adsorbed on alum by following the procedure of Example 2.

Example 4 The material from part B of Example 1 of Patent Specification No. 46793 , 1 ml, is added to 1 ml of a (v/v) solution of 2-mercaptoethanol in dionized water, and 1 ml of a 1% (v/v) solution of polyoxyethylene (20) sorbitan monooleate in deionized water . The resulting mixture is agitated gently and placed in a 37°C water bath. After 1 hour the mixture is diluted with SPGA using a previously calculated quantity of diluent until it contains 32 IAHA units per ml. The solution is then dispensed into plastic 2-ml screw-cap serum tubes (0.5 ml/tube) and stored in a liquid nitrogen freezer.

Claims (11)

1. A composition comprising hepatitis B core antigen in a medium consisting essentially of TMN-1% BSA as hereinbefore defined, saline or carbonate buffer.

2. A vaccine effective to produce hepatitis B core antibody comprising an inactivated composition according to Claim 1. .

3. Λ vaccine according to Claim 2 in which the inactivation is effected by an aqueous solution of formaldehyde .

4. A vaccine according to Claim 2 in which the medium is SPGA as hereinbefore defined.

5. A vaccine according to Claim 2 in which the core antigen is adsorbed on a physiologically acceptable substrate.

6. A vaccine according to Claim 4 in which the substrate is alum.

7. A method of preparing a vaccine effective to induce formation of antibody to hepatitis B core antigen that comprises inactivating a composition according to Claim 1.

8. A method according to Claim 7 in which the treated hepatitis B core antigen is adsorbed on alum.

9. A composition as claimed in Claim 1 substantially as hereinbefore described in Example 1.

10. A composition as claimed in Claim 2 substantially as hereinbefore described in Example 2 or 4.

11. A method as claimed in Claim 7 substantially as hereinbefore described in Example 3.